In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers, the track of the scanning light beam on or in the specimen ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). To allow the acquisition of image data in layers, the specimen stage or the objective is shifted after a layer has been scanned, and the next layer to be scanned is thus brought into the focal plane of the objective.
In many applications, specimens are prepared using a plurality of markers, for example several different fluorescent dyes. These dyes can be excited sequentially, for example with illuminating light beams that have different excitation wavelengths. Simultaneous excitation using an illuminating light beam that contains light of several excitation wavelengths is also common. European Patent Application EP 0 495 930 “Confocal microscope system for multi-color fluorescence,” for example, discloses an arrangement having a single laser emitting several laser lines. In practical use at present, such lasers are most often embodied as mixed-gas lasers, in particular as ArKr lasers.
Aberrations attributable to interference phenomena often occur in scanning microscopy. These interferences are usually caused by multiple reflections at various optical interfaces within the scanning microscope.
German Unexamined Application DE 100 42 114.8 A1 discloses a method for illuminating a specimen with light of a laser light source, preferably in a confocal scanning microscope. With the method, the coherence length of the laser light can be decreased so that troublesome interference phenomena in the image can be largely eliminated. If interference phenomena nevertheless do occur, they are to be influenced in such a way that they have no influence on detection. The method according to the invention is characterized in that the phase length of the light field is varied, using a modulation means, in such a way that interference phenomena in the optical beam path occur not at all, or only to an undetectable extent, within a definable time interval.
The method disclosed in the Unexamined Application is laborious in particular for high measurement speeds or images, and because of the special requirements in terms of illumination poses difficulties in terms of changing in the illuminating wavelength, or indeed illuminating with light containing multiple wavelengths.